Substituted 1, 3, 4, 10, 11, 12-hexahydroxynaphthacene-2-carboxamides



United States Patent 3,360,559 SUBSTITUTED 1,3,4,10,11,12-HEXAHYDROXY-NAPHTHACENE-Z-CARBOXAMIDES Jerry Robert Daniel McCormick, Spring Valley,and Elmer Raymond Jensen, Nanuet, N.Y., assignors to American CyanamidCompany, Stamford, Conn., a corporation of Maine No Drawing. Filed Nov.30, 1964, Ser. No. 414,899

8 Claims. (Cl. 260-559) ABSTRACT OF THE DISCLOSURE This disclosuredescribes compounds of the class of substituted 1,3,4,10,11,12hexahydroxynaphthacene-Z- carboxamides useful as intermediates in thesynthesis of physiologically active antibiotics of the tetracyclineseries.

This invention relates to new organic compounds and, more particularly,is concerned with the 4-hydroxy derivatives of substituted1,3,l(),11,12-pentahydroxynaphthacene-Z-carboxamides and to methods ofpreparing these novel compounds. The substituted 1,3,10,11,12-pentahydroxynaphthacene-Z-carboxamides are described in the copendingapplication of McCormick et al., Ser. No. 233,944, filed Oct. 29, 1962,now Patent No. 3,201,- 424. The novel compounds of the present inventionmay be represented by the following general formula:

R; Ra

CONH2 0H ()H OH )11 wherein R is hydrogen or lower alkyl, R is hydrogenor lower alkyl and R R and R are each selected from the group consistingof hydrogen, halogen, hydroxy, lower alkoxy, lower alkyl, amino,mono(lower alkyl)amino, di(lower alkyl) amino, nitro, nitroso, thiocyanoand mercapto. Lower alkyl and lower alkoxy groups contemplated by thepresent invention are those having from 1 to 6 carbon atoms. Halogen isexemplified by chlorine, bromine, iodine and fluorine.

A preferred embodiment of the present invention may be represented bythe following general formula: f i I CONE: 6H $11 11 in wherein Z ishydrogen or lower alkyl and Y is hydrogen, chlorine or bromine.

The novel compounds of the present invention are obtainable ascrystalline materials having characteristic absorption spectra and highmelting points. They are generally insoluble in water and most organicsolvents.

The novel compounds of the present invention may be readily prepared ina variety of different ways. For example, the condensation of anappropriately substituted 3-hydroxy phthalic anhydride with anappropriately substituted 2-carboxamido-l,3,4-trihydroxynaphthalenegives rise either to an intermediate 6-hydroxynaphthacene-5,12-

"ice

quinone or to an intermediate naphthacene-6,1l-quinone as set forth inthe following reaction scheme:

R1 R 0H 0 0 I I a- OH O m- CONH:

I CO I O H O R O H R1 0 H O OH I I II R 011 R CONH:

OH H i OH 9. 6-hydr0xynapthacene-5, 12-quinone R 0 R OH I II I RF OH I R-CON H a I l OH ii OH OH a naphthacene-fi, ll-quinone RF 0 H RB CONH:

I I OH OH OH O H R R 0 H R0 CONHz l I in OH OH OH wherein R R and R areas previously defined, and R is hydrogen or lower alkyl. When R ishydroxy or lower alkoxy, then the intermediate6-hydroxynaphthacene-5,12- quinone is obtained. When R is hydrogen orlower .alkyl, then the product is a naphthacene-6,ll-quinone. Thiscondensation is carried out under the general conditions of theFriedel-Crafts reaction employing, for example, boric anhydride,sulfuric acid, anhydrous aluminum chloride or anhydrous ferric chlorideas catalyst. The condensation may be carried out in a high boiling inertsolvent, or in a melt such as boric anhydride or the eutectic of sodiumchloride and aluminum chloride at temperatures in excess of C. Reductionof the resulting naphthacene-quinones to the corresponding5,6-deoxygenated derivatives may be'readily accomplished by any ofseveral well-known procedures described in the chemical literature forthe reduction of l-hydroxyanthraquinones to the corresponding9-anthrones. For example, this reduction may be accomplished withboiling hydriodic acid, with tin and hydrochloric acid, or by catalytichydrogenation with a nickel or noble metal catalyst.

Some of the novel compounds of the present invention may also beprepared by the condensation of an appropriately substituted3,7-dihydroxyphthalide with an appropriately substituted1,3,4-trihydroxynaphthalene-Z-carboxamide as set forth in the followingreaction scheme:

, R B1 \c 1 1, on B8 -o11 0 R OONH:

| 00 l 1 on on on R:- OH

RQ CONH:

on on on on I oo CONH2 & r

Ra OH R; CONH:

R7 OH Rs- OH R9 CONHZ OH OH OH OH wherein R R and R are as previouslydefined and R is hydrogen or lower alkyl. This condensation may also becarried out under the general conditions of the Friedel- Crafts reactionas described above, and reduction of the intermediate6-hydroxynaphthacene-5,l2-quinones may also be accomplished as set forthabove. In preparing the novel compounds of the present invention by thecondensation reactions set forth above, it is to be understood that thesubstituted phthalic acids, esters, and acid halides are equivalent tothe corresponding substituted phthalic anhydrides.

Certain of the novel compounds of the present invention may be preparedfrom an appropriately substituted 4 tetracycline methyl betaine[I.A.C.S. 80, 1654-1657 (1958)] as set forth in the following reactionscheme:

i CH3@NCH3 R1 0113 OH ll R9 VCONHfl I II II I! on o 0 R7 CHa OH OH Ra- Aon R9 OONH2 A) II H l Ilia (3H3 ()H H OH Rn CONH:

a l I H OH OH OH wherein R R and R are as previously defined. Theconversion of the tetracycline methyl betaine to the 4-dedimethylamino-4-hydroxy-4a,'lZa-anhydrotetracycl-ine is carried out inrefluxing acetonitrile under an atmosphere of nitrogen for 1 /2 to 2hours. After this reaction is complete, the solvent is evaporated todryness and the product is refluxed in a 30% solution of hydrobrornicacid in acetic acid whereby thel,3,4,10,11,12-hexahydroxynaphthacene-Z-car-boxamide is obtained.

The novel compounds of the present invention are useful as intermediatesin the synthesis of physiologically active antibiotics of thetetracycline series. For example, the substituted 1,3,4,l0,11,1Z-hexahydroxynaphthacene-2-carboxamides of the present invention maybe biologically transformed to tetracyclines as set forth in thefollowing reaction scheme:

Ilia Ra a OH R OH R CONH:

| I OH OH OH OH Rq OH 0E 55 ll wherein R R R R and R are as previouslydefined and R' is hydrogen, hydroxy or lower alkyl. This transformationis accomplished by adding the novel compounds of the present inventionto a fermentation medium inoculated with a strain of a species of thegenus Streptomyces which species is capable of producing one of thetetracyclines. Certain other biological transformations may beaccomplished simultaneously with the 5a,6- and 4a,12a-dihydration. WhereR in the above reaction scheme is hydrogen and a S-hydroxylating speciesof the genus Streptomyces is employed, then a hydroxyl group isintroduced at the 5-position. Where R is lower alkyl, or where R ishydrogen and a non S-hydroxylating species of the genus Streptomyces isemployed, then R',; is lower alkyl or hydrogen, respectively. Where R inthe above reaction scheme is a substituent other than hydro-gen, thenthis substituent is retained at the 7-position regardless of thebiological transformations occurring in the rest of the molecule. WhereR is hydrogen, and a non-halogenating strain of Streptomyces isemployed, then R, is also hydrogen in the product. Where R in themolecule is hydrogen, and a 7-halogenating strain of the genusStreptomyces is employed, then R in the product is chlorine or brominedepending upon the conditions of the fermentation. Among the strains ofS. aureofaciens which will introduce chlorine or bromine at the7-position of the molecule, as well as accomplishing the 5a,6- and4a,12a-dihydration, are the following:

S. aureofaciens ATCC 10762a ATCC 12554 ATCC 10762b ATCC 13189 ATCC10762g ATCC 13899 ATCC 10762i ATCC 13900 ATCC 11989 NRRL B-1286 ATCC12416b NRRL B-1287 ATCC 12416c NRRL B1288 ATCC 124 1 6d NRRL B-2209 ATCC12551 NRRL B2406 ATCC 12552 NRRL B2407 ATCC 12553 NRRL 3013 Arepresentative strain of the genus Streptomyces which is anon-halogenating strain, that is, which will not introduce halogen atthe 7-position of the molecule, but which will accomplish the 5a,6 and4a,12a-dihydration, is S. aureofaciens NRRL 3014. Represenative strainsof the genus Streptomyces which are non-halogenating strains but whichwill introduce a hydroxy group at the 5-position of the molecule, inaddition to accomplishing the 5a,6- and 4a,12a-dihydration, are S.rimosus NRRL 2234, S. platenis NRRL 2364 and S. hygroscopicus NRRL 3015.

The conditions of the fermentation for the biological conversion of thecompounds of the present invention to tetracyclines are generally thesame as set forth in US. Patent 2,482,055 to Duggar, US. Patent2,734,018 to Minieri et al. and US. Patent 2,878,289 to McCormick et al.and which, in turn, are generally the same as for the presently knownmethods for producing various tetracyclines by fermentation. That is,the fermentation medium contains the usual nutrients and mineralsubstances. Suitable nutrients include any assimilable source of carbon,such as the polysaccharides or starches, or polyalcohols such asglycerol may be used. An assimilable source of nitrogen may be suppliedthrough the use of proteins, protein hydrolysates, urea, corn steepliquor, meat extracts, peptone, distillers solubles, fish meal and otherconventional substances. The common anions and cations are supplied inthe form of their non-toxic salts. Trace elements such as manganese,cobalt, zinc, copper, etc., are obtained either as impurities in theabove compounds, or through the use of tap water or by specificallyadding solutions especially enriched with these trace elements.

The other general conditions of the fermentation such as hydrogen ionconcentration, temperature, time rate of aeration, preparation of theinoculum, sterilization, inoculation and the like are conventional andare similar to those for the production of other tetracyclines as setforth in the aforementioned US. Patents to Duggar, Minieri et al. andMcCormick et a1.

When a 7-helogenating strain of the genus Streptomyces is employed witha 1,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamide wherein R ishydrogen, it is necessary only to modify the fermentation medium so thatit contains at least 10 parts per million of chloride ions when the7-chloro substituent is desired, or a like amount of bromide ions whenthe 7-bromo substituent is desired.

After the fermentation has been continued for a suitable time, forexample, from 12 to 96 hours, and the transformation of the compound tothe desired tetracycline is substantially complete, the tetracyclineproduct may be isolated from the fermentation mash in any convenientmanner. The isolation process may be selected from any of the numerousisolation techniques now well known in the art.

The starting material may be added at any desired concentration,although for practical reasons a substrate at a concentration of up toabout 10 grams per liter of medium is satisfactory although higherconcentrations may be used with some sacrifice in yield. The addition ofthe starting material may be accomplished in any suitable manner so longas it promotes contact with the biological medium. To this end, it ispreferred to add the starting material in a solvent such asdimethylforrnamide, dimethylacetamide, dimethylsulfoxide,tetramethylenesulfoxide and N-methylpyrrolidone. However,dimethylsulfoxide is preferred and a solution of magnesium acetate indimethylsulfoxide is the most preferred solvent for the startingmaterial. Solutions of the1,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamides must be protectedfrom air as the compounds are readily oxidized in solution.

The invention will be described in greater detail in conjunction withthe following specific examples.

EXAMPLE 1 Synthesis 0] 1,3, 1,10,11,IZ-hexahydroxynaphthacene- 2-carboxamide EXAMPLE 2 Biological conversion of1,3,4,10,1LIZ-hexahydroxynaphthacene 2 ciw-boxamide to7-chlor0-6-demethyltetrw cycline Spores of S. aureofaciens NRRL 3013were washed from an agar slant with sterile distilled water to form asuspension containing 6080 10 spores per ml. A 0.33 ml. portion of thissuspension was used to inoculate an 8 inch test tube containing 8 ml. ofa medium prepared according to the following formulation:

Sucrose grams 30 Ammonium sulfate do 2 Calcium carbonate do 7 Cornsteepliquor do 20 Tap water, q.s. to "milliliters" 1000 Prior to inoculation,the medium was sterilized by autoclaving for 20 minutes under a pressureof 15 pounds per square inch. The inoculated tube was then incubated for24 hours at 28 C. on a reciprocating shaker operating at 116oscillations per minute, whereby an inoculum of the S. aureofaciens wasobtained. A fermentation medium of the following compositions wasprepared:

(NH SO grams 6.7 CaCO do 9.0 CoCl 6H O milligrams 5.0

NH C1 grams 2.0

7 MnSO (70% Technical Grade) do 0.10 Cornsteep liquor do 25.0 Starch do52.5 Corn flour do 14.5 Tap water, q.s. to -1 milliliters 1000 Aftersterilization of this medium in an autoclave for 20 minutes at apressure of 15 pounds per square inch, a 25 ml. portion in a 250 ml.Erlenmeyer flask was inoculated with a 1.0 ml. portion of the S.aureofaciens inoculum. The fermentation was carried out at 25 C.

for 24 hours on a rotary shaker operating at 180 revolu- EXAMPLE 3Preparation of 4-dedimethylamin0-4-hydr0xy-4a,12aanhydrotetracycline A2.0 gram portion of tetracycline methyl betaine was suspended in 500 ml.of acetonitrile. Nitrogen was bubbled through this suspension for 15minutes. The suspension was then heated under nitrogen atmosphere to theboiling point of acetonitrile and refluxed for 1 /2 hours. The heat wasthen removed and the bubbling of nitrogen was continued for anadditional 15 minutes until allof the trimethylamine vapors had beenstripped off. An orange crystalline product precipitated upon cooling.This precipitate was collected by filtration, washed with acetonitrileand dried in a vacuum desiccator to afford 100 mg. of product.

EXAMPLE 4 Conversion'of 4-a'edimelhylamin0-4-hydr0xy4a,12a-anhydrotetracycline to 6-methyl-1,3,4,10,11,12hexahydroxynaphthacene-Z-carboxamide A 100 mg. portion of4-dedimethylaminoi-hydroxy- 4a,12a-anhydrotetracycline was suspended in15 ml. of a solution of 30% hydrobromic acid in acetic acid. Thissuspension was heated at 50-55 C. for 3 hours. The suspension was cooledto room temperature and the precipitate was collected by filtration,washed with water and dried in a vacuum desiccator for 16 hours,affording 89 mg. of product.

EXAMPLE 5 Biological conversion of 6methyl-1,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamide to 7chlorotetracycline Spores of S. aureofaciens NRRL 3013 were washed froman agar slant with sterile distilled water to form a suspensioncontaining 60 million to 80 million spores per ml. A 0.33 ml. portion ofthis suspension was used to inoculate an 8 inch test tube containing 8ml. of an inoculum prepared as described in Example 2. A fermentationmedium of the following composition was prepared:

(NH SO grams 6.7 CaCO d0 9.0 CoCl -6H O milligrams 5.0 NH Cl grams 2.0MnSO (70% Technical Grade) do 0.10 Cornsteep liquor do 25.0 Starch do52.5 Corn flour do 14.5 Tap water, q.s. to milliliters 1000 Aftersterilization of this medium, 25ml. portions in 250 ml. Erlenmeyerflasks were inoculated with 1 ml. portions of the S. aureofaciens NRRL3013 inoculum. The fermentation was carried out at 28 C. for 24 hours ona rotary shaker operating at revolutions per minute. At this time eachmash portion was transferred to an individual flask containing asolution of 10.7 mg. of 6- methyl-1,3,4,l0,11,l2-hexahydroxynaphthacene2 carboxamide in a mixture of 30 mg. of magnesium acetate and 1 ml. ofdimethylsulfoxide. The fermentation was continued on the rotary shakerfor an additional 96 hours at 28 C. At this time, biological assays ofthe mash indicated the presence of antibacterial activity correspondingto 264 micrograms of 7-chlorotetracycline per ml. This corresponds to ayield of 49% based on the compound added. The identity of the product as7-chlorotetracycline was confirmed by paper chromatography in abutanol-pH3 phosphate buffer system. A control flask run in the samemanner but with theaddition of only 30 mg. of magnesium acetate and 1ml. of dimethylsulfoxide and no6-methyl-l,3,4,10,11,12-hexahydroxynaphthacene-2-carboxamide, showed no7-chlorotetracycline.

EXAMPLE 6 Preparation of 6-demethyl-7-chlorotetracycline methiodia'e The6-demethyl-7-chlorotetracycline free base, 3 g., is dissolved in 60 ml.of hot tetrahydrofuran and the solution is filtered. To the filtrate isadded 9 ml. of methyl iodide and the mixture is allowed to stand at roomtemperature for 6 days. A small amount of amorphous precipitate isfiltered, washed with ether and dried to afford 396 mg. of product. Thefiltrate is diluted with about 200 ml. of ether and the light yellowsolid is isolated by filtration. Yield: 3.18 g. This material iscrystallized from methanol as follows: The solution of 1 g. of productin 4 ml. of methanol is stirred at room temperature for 10 minutes.Crystalline precipitate is filtered and dried in pistol at 62 C. for twohours. This product has the following properties:

Mgfif =228 my [log 6 4.41], 270 my [log 6 4.14], 373

my [log e 4.03]

A9 51? """=225 m [log e 4.41], 290 my [log 6 4.09], 375 my [log e 4.20]

EXAMPLE 7 Preparation of 6-demethyl-7-chlorotetracycline methyl betaineThe 6demethyl-7-chlorotetracycline methiodide, 200 mg., is dissolved in5 ml. of water. The solution is adjusted immediately to pH 6.8 with afew drops of 20% sodium acetate solution. One ml. of methanol is addedto get the precipitated solid into solution which is then stirred for 10minutes at room temperature. Methanol is stripped off under vacuum andthe crystalline product which precipitates from water is filtered anddried at 62 C. overnight to afford 143 mg. of product.

EXAMPLE 8 Preparation of4-dedimethylamino-4-hydroxy-4a,12aanhydr0-6-demethyl-7-chlorotetracyclineThe 6-demethyl-7-chlorotetracycline betaine, 3 g., is suspended in 500ml. of acetonitrile and nitrogen is bubbled through for 15 minutes. Thesuspension is then heated under nitrogen atmosphere to the boiling pointof acetonitrile and refluxed for 4 hours. A small amount of solid doesnot go in solution. Heat is removed and bubbling of nitrogen iscontinued until most of the trimethylamine vapors are stripped off(about 10 minutes). The undissolved material is filtered. The filtrateis evaporated to dryness to afford 2.1 g. of a mixture which is purifiedas follows: 1.5 g. of this material is dissolved 9 in 500 ml. ofmethanol. A yellow product crystallizes out within a few minutes. It isallowed to stand at room temperature for one hour and then filtered. Itis washed well with ether and dried in pistol at 62 C. Yield: 381 mg.

k11 i: :285 my [log 6 3.86], 400 m [log 6 4.05]

Preparation of7-chl0r0-1,3,4,10,1LIZ-hexahydroxynaphthacene-Z-carbaxamide 4.61], 481my. [log 6 4.16], 534 in [log 6 3.96]

EXAMPLE Preparation of4-dea'imethylamin0-4-hydroxy-4a,12aanhydro-7-chlorotetracycline The7-chlorotetracycline methyl betaine, 3.44 g., is suspended in 630 ml. ofacetonitrile and nitrogen is bubbled through for minutes. The suspensionis then heated under nitrogen atmosphere to the boiling point ofacetonitrile and the resulting solution is refluxed for one and a halfhour. Heat is removed and bubbling of nitrogen is continued until mostof trimethylamine vapors are stripped off (about 10 minutes). A smallamount of solid precipitates out upon cooling which is filtered off. Thefiltrate is evaporated to dryness. Solid is received in 500 ml. of etherand refluxed for 30 minutes. Undissolved solid is filtered (1.713 g.).Filtrate Which contains the desired products is evaporated to dryness(wt.=1.192 g.) and purified by partition column chromatography using thesystem: heptane-ethyl acetate-methanol-water (80-20- 174). The productis obtained in two tautomeric forms which give the same ultravioletspectra in 0.1N HCl as follows: A 250 m [log 6 4.32], 362 m [log e3.90].

EXAMPLE 11 Preparation of 7-chI0r0-6-methyl-1,3,4,10,1],12-

hexahydroxynaphfhacene-Z-carboxamide The 4dedimethylamino-4-hydroxy-4a,12a-anhydro-7- chlorotetracycline, mg., isdissolved in 4 ml. of a solution of 32% hydrobromic acid in acetic acidand the red solution is evaporated immediately to dryness under vacuum.The solid is received in ether, filtered and dried in vacuo to afford16.7 mg. of product analyzing as follows: k =270 [10g 6 4.32], 310 [log5 4.39], 508 [log 6 3.98]. The spectrum is taken in concentrated H 50containing 1% sodium borate.

EXAMPLE 12 Biological conversion of 7-chl0r0-6-methyl-1,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamide to 7-chl0r0- tetracycline utilizinga non-chlorinating strain of S. aureofaciens Spores of non-chlorinatingS. aureofaciens NRRL 3014 were washed from an agar slant with steriledistilled water to form a suspension containing 60-80 10 spores per ml.A 0.33 ml. portion of this suspension was used to inoculate an 8 inchtest tube containing 8 ml. of a medium prepared according to thefollowing formulation:

Sucrose grams 30 Ammonium sulfate do 2 Calcium carbonate do 7 Cornsteepliquor do 20 Tap water, q.s. to mi1liliters 1000 Prior to inoculation,the medium was sterilized .by autoclaving for 20 minutes under apressure of 15 pounds per square inch. The inoculated tube was thenincubated for 24 hours at 28 C. on a reciprocating shaker operatin g at116 oscillations per minute whereby an inoculum of the S. anreofacienswas obtained. A fermenation medium of the following composition wasprepared.

(NH SO grams 6.7 CaCO do 9.0 CoCl 6H O "milligrams" 5.0 NH CI grams 2.0MnSO (70% Technical Grade) d0 0.10 Cornsteep liquor do 25.0 Starch do52.5 Corn flour do 14.5 Tap water, q.s. to milliliters 1000 Aftersterilization of this medium in an autoclave for 20 minutes at apressure of 15 pounds per square inch, 25 ml. portions in 250 m1.Erlenmeyer flasks were inoculated with 1.0 m1. portions of the S.aureofaciens inoculum. The fermentation was carried out at 25 C. for 24hours on a rotary shaker operating at 180 revolutions per minute. Atthis time each mash portion was transferred to an individual flaskcontaining a solution of 10 mg. of 7-chl0ro-6-methyl 1,3,4,10,11,12hexahydroxynaphthacene-2-carboxamide in a mixture of 10 mg. of magnesiumacetate and 1 ml. of dimethylsulfoxide. The fermentation was thencontinued on the rotary shaker for an additional 96 hours at 28 C. Atthis time analysis of the mash showed the presence of 10 micrograms perml. of 7-chlorotetracycline. A control flask run in exactly the samemanner but omitting the 7-chloro-6-methyl-1,3,4,10,11,12-hexahydroxynaphtha cene-2-carboxamide showed no 7- chlorotetracycline.

EXAMPLE 13 Biological conversion of 7-chl0r01,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamide to7-chl0ro-6-demethyltetracycline utilizing a non-chlorinating strain ofS. aureofaciens The procedure of Example 12 was followed with theseexceptions: The partially fermented (24 hour) mash was transferred toflasks containing a solution of 10 mg. of 7-chloro-1,3,4,10,11,12hexahydroxynaphthacene-Z-carboxamide in a mixture of 10 mg. of magnesiumacetate and 1 ml. of dirnethylsulfoxide. The fermentation was thencontinued on the rotary shaker for an additional 96 hours at 28 C. Atthis time, analysis of the mash showed the presence of 9 micrograms perml. of 7-ch1oro- G-demethyltetracycline. A control flask run in exactlythe same manner but omitting the 7-chloro-1,3,4,l0,11,12-hexahydroxynaphthacene-2-carboxamide showed no 7-ch'loro-6-demethyltetracycline.

EXAMPLE l4 Biological conversion of 7-chl0r0-6-methyl-1,3,4,10,11,

1Z-hexahydroxynaphthacene-Z-carboxamide to 7 -chl0r0- tetracyclineutilizing a chlorinating strain 0 S. aureofaciens The procedure ofExample 12 was followed with these exceptions: Spores of chlorinating S.aareofaciens NRRL 3013 were Washed from an agar slant with steriledistilled water to form a suspension containing 6 0 10 spores per ml. A0.33 ml. portion of this suspension was used to inoculate an 8 inch testtube containing 8 ml. of the medium described in Example 12. Thepartially fermented (24 hour) mash (prepared as in Example 12) wastransferred to flasks containing a solution of 10 mg. of7-chloro-6-methyl-1,3,4,10 ,11,12 h'exahydnoxynaphthacene-Z-carboxamidein a mixture of 10 mg. of magnesium acetate and 1 ml. ofdimethylsulfoxide. The fermentation was then continued on the rotaryshaker for an additional 96 hours at 28 C. At this time, analysis of themash showed the presence of 10 micrograms per EXAMPLE 15 Biologicalconversion of 7-chl0r0-1,3,4,10,11,12-hexahydroxynaphthacene-Zcarbo'xamide to 7-chl0r0-6-demethyltetracycline utilizing a chlorinatingstrain S. aureofiaciens The procedure of Example 14 was followed withthese exceptions: The partially fermented (24 hour) mash was transferredto flasks containing a solution of 10' mg. of 7 chloro-1,3,4,10,11,12hexahydro-xynaphthacene-Z-carboxamide in a mixture of 10- mg. ofmagnesium acetate and 1 ml. of dimethylsulfoxide. The fermentation wasthen continued on the rotary shaker for an additonal 96 hours at 28 C.At this time analysis of the mash showed the presence of 9 microgramsper ml. of 7-chloro-6-demethyltetracycline. A control flask run inexactly the same manner but omitting the7-chloro-l,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxarnide showed no7-chloro-6- demethyltetracycline.

What is claimed is:

1. A compound of the formula:

e f: (])H Rtr- OH RF- CONH2 $11 ()11 OH ()H wherein R is selected fromthe group consisting of hydrogen and lower alkyl, R is selected from thegroup consisting of hydrogen and lower alkyl, and R R and R are eachselected from the group consisting of hydrogen, halogen, hydroxy, loweralkoxy, lower alkyl, amino, mono(lower alkyDamino, di(loWer alkyl)amino,nitro, nitroso, thiocyano and mercapto.

2. A compound of the formula:

OH OH OH OH wherein Z is selected from the group consisting of hydrogenand lower alkyl, and Y is selected from the group consisting ofhydrogen, chlorine and bromine.

3. 1,3,4,10,11,12 hexahydroxynaphthacene-Z-carhoxamide.

4. 6 Inethyl-l,3,4,10,11,1Z-hexahydroxynaphthacene- Z-carboxamide.

5. 7 chloro-1,3,4,10,ll,l2 hexahydroxynaphthacene- 2-carboxamide.

6. 7 chloro-6-rnethyl-1,3,4,10,11,12hexahydroxynaphthacene-2-carboxamide.

7. 7 bromo-l,3,4,10,11,1Z-hexahydro-xynaphthacene- 2-carboxamide.

8. 7 bromo-6-methyl-1,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamide.

References Cited FOREIGN PATENTS 1,090,205 10/1960 Germany.

NICHOLAS S. RIZZO, Primary Examiner.

1. A COMPOUND OF THE FORMULA: